2.
NRG, NMDARs, and Activity Regulate CNS Myelination
cells, making these cells more sensitive to glutamate released from
active axons. As a result, this interaction of NRG and
glutamatergic signalling accelerates and increases myelination,
but most importantly provides a mechanism by which myelination
is focussed on the axons of active neurons. Investigations of white
matter pathology based on this finding revealed that remyelination
of axons in a demyelinated lesion in vivo is NMDA receptor
dependent, suggesting enhancement of NRG- and NMDARdependent remyelination as a therapeutic approach for promoting
recovery after demyelination.
Author Summary
Myelination acts as an insulator for neurons and as such is
essential for normal brain function, ensuring fast neuronal
communication. Oligodendrocytes are the cells that wrap
their membrane around nerve cell axons to form the
myelin sheath that enables fast action potential propagation. However, what determines whether an individual
axon becomes myelinated remains unknown. We show
that there are two distinct modes of myelination: one that
is independent of neuronal activity and the release of the
neurotransmitter glutamate and another that depends on
nerve cell action potentials releasing glutamate, which
then activates a class of glutamate receptor (NMDA
receptors) on oligodendrocyte lineage cells. We find that
the protein neuregulin switches oligodendrocytes between these two modes of myelination; neuregulin
increases oligodendrocyte lineage cells’ sensitivity to
glutamate by increasing the current flowing through their
glutamate receptors. With neuregulin present, myelination
is accelerated and increased. Blocking NMDA receptors
reduces the amount of myelination to far below its level
without neuregulin. Thus, a neuregulin-controlled switch
enhances the myelination of active axons. We also
demonstrate that remyelination after white matter damage (as occurs in diseases, such as spinal cord injury and
multiple sclerosis) is NMDA receptor-dependent. These
data help us understand the signalling that regulates
myelination and suggest the possible involvement of
neuregulin in schizophrenia and in remyelination after
white matter damage.
Results
NRG Increases Myelination by Oligodendrocytes
To assess whether NRG-ErbB signalling and activation of
NMDA receptors in oligodendrocytes interact to control myelination, we studied myelination of cultured dorsal root ganglion
(DRG) neurons by forebrain oligodendrocytes [8]. This allows
more detailed investigation of the underlying signalling mechanisms than is possible in transgenic studies where compensation for
gene knockout may occur (see Discussion). In this coculture
system, compact myelin is produced (Figure S1A) [8] with a
lamellar repeat distance of 10.961.8 nm (n = 6) and a g-ratio of
0.7460.02 (n = 6), as expected for CNS myelination [26,27].
Myelinating and nonmyelinating oligodendrocytes are distinguishable after labelling for MBP and neurofilament (NF) (Figure 1A,B).
Myelinating oligodendrocytes enwrap NF-expressing axons with
MBP-expressing processes, at the lateral ends of which the
paranode labels for the axon-oligodendrocyte junction protein
Caspr (Figure S1B). When two oligodendrocytes myelinate
adjacent segments of the same axon, a node of Ranvier is
identifiable by the presence of Caspr labelling flanked by MBP
labelling on either side of the node (Figure S1C). Myelination does
not occur in the absence of added OPCs, it is mediated purely by
OPCs and not by Schwann cells, and only DRG neurons and not
interneurons become myelinated (Figure S1D–F).
ErbB signalling was activated by applying the extracellular
domain of NRG (NRG1 type 1-b1, 10 ng/ml, 0.33 nM), which
includes the EGF domain that binds to ErbB receptors expressed
on oligodendrocytes [28] and mimics [3] the effect of membranebound NRG. As found previously [8], adding NRG increased the
fraction of oligodendrocytes that myelinated axons (Figure 1C,E,
Figure 2A). Myelination depends on the local axon density [8], so
we quantified myelination (3 wk after plating OPCs on the
neurons) as the slope of a plot of the fraction of oligodendrocytes
that were myelinating against local axon density [8] (see
Figure 1C–F, Figure 2A, and Materials and Methods; similar
results were obtained with other quantification methods, see
Figures S2 and S3). Measured in this way, adding NRG increased
myelination by 42% (p = 761024; Figure 2A). NRG also increased
MBP protein expression in the cocultures (p = 0.03; Figure S1G),
without affecting MBP or MOG mRNA levels (Figure S1H),
indicating a local posttranscriptional regulation of these myelin
genes [16].
Varying the time at which myelination was assessed after plating
the OPCs onto the neurons showed that NRG both accelerated
myelination and increased its steady state level (Figure 2C). The
myelination at steady-state level was 16% higher (p = 0 .024), and
its onset (defined as the time to reach half the maximal value)
occurred 5 d earlier (p = 0.0037), according to the fitted curves in
Figure 2C.
To assess whether changes in cell proliferation, growth, or
survival were responsible for the increased myelination in NRG,
we counted cells of different types identified with antibodies. In the
there is controversy over its actions. In the peripheral nervous
system, a decrease of NRG signalling leads to less myelination
[3,4]. Decreasing NRG-ErbB signalling has also been reported to
reduce myelination by oligodendrocytes in the central nervous
system [5–8]. Contradicting this, however, knocking out NRG or
ErbB was found to have no effect on myelination, although
overexpressing NRG increased myelination [9].
Further uncertainty relates to how neuronal activity regulates
CNS myelination. Neuronal activity can promote myelination
[10,11], yet oligodendrocytes can ensheath dead axons that lack
any activity [12]. An activity dependence to myelination could
reflect action potential evoked release of NRG [13], but glutamate
is also released onto oligodendrocyte precursor cells by action
potentials in unmyelinated axons [14–17] and could potentially
initiate myelination. Glutamate activates both AMPA/kainate and
NMDA receptors in oligodendrocyte lineage cells [14–18], and the
presence of NMDA receptors in oligodendrocyte processes [18–
20] is consistent with them having a role in myelination.
Furthermore, glutamate signalling and NRG signalling might
interact to control myelination, since, in the grey matter at least,
NRG increases the expression of NMDA receptor subunits in
neurons [21] and in forebrain [22]. However, contradictory data
have been presented on the contribution of NMDA receptors to
CNS myelination, with suggestions that they either play no role
[23,24] or that their activation by glutamate released from axons
promotes myelin basic protein (MBP) expression and myelination
[16,25].
Here we show for the first time that there are two distinct modes
of myelination by oligodendrocytes, one independent of neuronal
activity and the other dependent on neuronal action potentials.
Furthermore, we demonstrate that NRG switches oligodendrocytes between these two myelination programmes by increasing
NMDA receptor-mediated currents in oligodendrocyte lineage
PLOS Biology | www.plosbiology.org
2
December 2013 | Volume 11 | Issue 12 | e1001743

3.
NRG, NMDARs, and Activity Regulate CNS Myelination
A Myelinating oligodendrocyte
B Non-myelinating oligodendrocyte
MBP NF
MBP NF
20 μm
▲
MBP NF
▲
▲
▲
100 μm
MBP NF
0.8
0.6
▲
0.4
0.2
0.0
0
▲
▲
▲
D MK801
1.0
20
40
100 μm
60
fraction of OLs myelinating
C Control
fraction of OLs myelinating
20 μm
1.0
0.8
0.6
0.4
0.2
0.0
0
▲
▲
100 μm
0.8
0.6
▲
▲
MBP NF
0.4
▲
0.2
0.0
0
100 μm
20
40
60
fraction of OLs myelinating
▲
F Neuregulin + MK801
1.0
▲
MBP NF
fraction of OLs myelinating
E Neuregulin
20
40
1.0
0.8
0.6
0.4
0.2
0.0
0
axonal density, %
20
40
PLOS Biology | www.plosbiology.org
NRG’s Effect Depends on Action Potentials
To test the role of neuronal activity in regulating myelination we
applied TTX (1 mM) to block action potentials. TTX had no effect
on myelination in the absence of added NRG (p = 0.65), but
reduced myelination by 50% in the presence of NRG (p = 0.006;
Figure 2A)—that is, TTX abolished the increase in myelination
produced by NRG (both when added at the same time as NRG
and when added 3 d later; Figure S5A). Thus, whereas in the
absence of NRG myelination occurs predominantly by a
3
60
axonal density, %
Figure 1. Effect of NRG and NMDA receptor block on myelination. (A, B) High-magnification views of a myelinating oligodendrocyte (A) with
MBP (green) expressed in processes wrapping around axons expressing NF 160/200 (NF, red), and of a nonmyelinating oligodendrocyte (B) with MBP
expressed (in a more patchy and often diffuse manner) in processes that are not aligned with axons. Myelination was quantified as the fraction of all
MBP-expressing oligodendrocytes that provided a thick straight myelin sheath to at least one axon. (C–F) Myelinating processes (MBP, green)
wrapping DRG axons (NF, red) in control conditions (C), in the presence of MK-801 (D), in the presence of NRG (E), and in the presence of NRG and
MK-801 (F). Filled and open arrows show some myelinating and nonmyelinating oligodendrocytes. Graphs show fraction of oligodendrocytes that are
myelinating, versus fraction of area occupied by DRG processes, for 30 images of each coverslip from which the specimen images shown were taken,
best fit with a linear dependence of myelination on axon density.
doi:10.1371/journal.pbio.1001743.g001
presence of NRG, there was no significant change in the density of
axons (Figure 2B), the total number of DAPI-labelled cells
(Figure 3A), the number of MBP-labelled oligodendrocytes
(Figure 3B), or the percentage of cells that were oligodendrocyte
lineage cells, OPCs, interneurons, astrocytes, or satellite cells
(Figure 3C–G) in the cocultures. No microglia were detected using
anti-CD11b antibody or isolectin B4. Thus, the effect of NRG is
not due to an altered cell density or changes in environmental
paracrine signals or trophic factors caused by altered cell numbers.
60
axonal density, %
axonal density, %
December 2013 | Volume 11 | Issue 12 | e1001743

8.
NRG, NMDARs, and Activity Regulate CNS Myelination
resistance of oligodendrocyte lineage cells was not altered (OPCs,
control 8366369 MV versus NRG 8946483 MV, p = 0.92;
differentiated oligodendrocytes, control 130625 MV versus
NRG 208665 MV, p = 0.20). However, with added NRG the
NMDA-evoked currents at 264 mV in oligodendrocyte precursor
cells and in differentiated oligodendrocytes were ,6-fold larger
than in cells not exposed to added NRG (p = 0.02 and p = 0.03,
respectively; Figure 5G–J). In contrast, kainate-evoked currents
(mediated by AMPA/kainate receptors) were not significantly
affected by NRG in either OPCs (p = 0.48) or differentiated
oligodendrocytes (p = 0.73; Figure 5G–J). NRG did not significantly alter the size of NMDA- or kainate-evoked currents in DRG
neurons (p = 0.75 and p = 0.93, respectively; Figure 5K), interneurons (p = 0.30 and p = 0.92; Figure 5L), astrocytes (p = 1 and
p = 0.78; Figure 5M), or satellite cells (p = 0.62 and p = 0.52;
Figure 5N) that were also present in the cultures. These data
indicate that NRG specifically upregulates NMDA responses in
oligodendrocyte lineage cells, providing a mechanism for these
cells to become more sensitive to glutamate released from active
axons.
NMDA receptors in oligodendrocyte lineage cells have been
suggested to mainly comprise NR1, NR2C, and NR3A subunits
[18–20,29]. NR2B and NR2C are the main subunits known to
be phosphorylated (by Fyn and Akt, respectively) downstream of
NRG and other growth factor signalling [30,31], and NR2C
phosphorylation promotes subunit trafficking to the plasma
membrane [31]. We therefore investigated whether the increase
in NMDA responses reflected altered receptor subunit expression
or phosphorylation, focussing on the possible role of NR2B,
NR2C, and NR3A subunits (in fact, no NR2A or NR2D and
very little NR3B were detected in the cocultures). Western
blotting revealed unchanged levels of NR1, of NR2B and its
phosphorylated form, of NR2C and its phosphorylated form,
and of NR3B (Figure 6A,B). However, the level of NR3A protein
was down-regulated by 40% in the presence of NRG (p = 0.02;
Figure 6A,B). NRG did not affect the NR3A protein level in
pure DRG cultures (p = 0.85; Figure 6C), but downregulated
NR3A by 33% in pure OPC cultures provided that glutamate
was also added to mimic glutamate release from DRG axons
(p = 0.02; Figure 6D and see Discussion). Removal of NR3A
subunits from NMDA receptors composed of NR1, NR2, and
NR3 subunits has previously been reported to increase their
single channel current, their trafficking to the surface membrane,
and their calcium permeability, and thus to increase NMDAevoked currents 2.8-fold [29,32,33]. Consequently, a downregulation of NR3A synthesis or an increase of its degradation
could account for the increased NMDA receptor current seen in
NRG.
Signalling Underlying the Effects of NRG
NRG can activate the PI3K-Akt or MAPK/Erk pathways in
oligodendrocytes [37]. To test which pathway mediated NRG’s
effects, we measured the levels of phosphorylated Akt and Erk in
the cocultures. NRG increased Akt phosphorylation by 43%
(p = 0.047; Figure 7B), with no effect on Erk phosphorylation
(p = 0.79; Figure 7C). In contrast in pure DRG cultures neither
pAkt nor pErk levels were affected (Figure S5B,C). To test whether
activation of Akt was downstream of the increased NMDA
receptor activation produced by NRG, we measured the levels of
pAkt and pErk in cocultures treated with MK-801 (Figure 7B,C).
MK-801 had no effect on the actions of NRG on Akt: NRG added
with MK-801 increased pAkt (p = 0.01) with no effect on pErk
(p = 0.26), and there was no significant difference between the
levels of pAkt in NRG, with or without MK-801 (p = 0.50). Thus,
activation of Akt does not occur downstream of NMDA receptor
activation (it may be upstream of the NRG-evoked increase in
NMDA receptor current, or on an independent NRG-activated
pathway). NRG also increased by 65% (p = 0.02) the proportion of
Nkx2.2-expressing cells (late OPCs/immature oligodendrocytes)
that expressed phosphorylated CREB (pCREB), an Akt target
[38], without affecting the percentage of cells lacking Nkx2.2 that
expressed pCREB (p = 0.35; Figure 7D,E).
Next we looked into CREB target genes. NRG increased more
than 4-fold the message level for the early growth response gene
Egr-1 (p = 0.036; Figure 7F), a CREB target gene [39] that has
been shown to be regulated by integrin b1-dependent PI3K/Akt
activation [40], neuronal activity [41], activation of NMDA
receptors [42], and activation of OPC glutamate receptors [43].
EGR-1 is an upstream regulator of MRF [44], an essential
transcription factor needed for myelination [45]. NRG more than
doubled the level of the CREB target c-Fos, but this did not reach
significance (p = 0.06) and had no effect on the level of c-Jun
(p = 0.36; Figure 7F).
These data suggest that NRG promotes myelination via Akt,
integrin b1, NMDA receptors, CREB, and EGR-1 signalling. This
is consistent with constitutively active Akt promoting myelination
[46], with b1 integrin activating Akt to promote myelination [34],
and with CREB activating transcription of myelin genes [39,47].
BDNF Also Induces NMDAR-Dependent Myelination
The role of NRG/ErbB signalling in CNS myelination is
controversial, as decreasing NRG or ErbB function reduced
myelination [5–7], while knocking NRG or ErbB out had no effect
on myelination [9]. Because of the importance of myelination,
there may be redundancy in the mechanisms regulating it. Like
NRG, BDNF increases NMDA receptor currents in neurons by
upregulating NR2C subunits [48], and via TrkB it can promote
oligodendrocyte differentiation, expression of MBP [49], and CNS
myelination [50]. We therefore tested the effect of adding BDNF
(10 ng/ml, 0.7 nM) to the cocultures. BDNF increased myelination (by 102%, p = 0.006; Figure 7G) and MBP expression
(p = 0.048; Figure S1G) and, as for NRG, made myelination
become dependent on activation of NMDA receptors (Figure 7G).
With BDNF present, MK-801 reduced myelination by 76%
(p = 6.961024), to only 47% of its value without BDNF (p = 0.019).
Like NRG, BDNF induced phosphorylation of Akt but not Erk
(Figure 7H–I; p = 0.03 and p = 0.2, respectively), and downregulated expression of the NR3A subunit but not the NR1 subunit of
NMDA receptors (Figure 7J; p = 0.005 and p = 0.69, respectively).
Thus, by acting through similar signalling pathways, BDNF can
also switch oligodendrocyte myelination to a pathway that
depends on glutamate release activating NMDA receptors.
Consequently, when the NRG/ErbB pathway is knocked out,
Only Activity-Dependent Myelination Is Integrin
Dependent
Myelination depends on integrins [34], and integrins modulate
NMDA receptor expression [35]. We therefore tested whether the
NRG-evoked switch from activity-independent to NMDA receptor-dependent myelination depends on integrins, using an
antibody to b1 integrin (1 mg/ml) to block its function [36]. This
had no effect on myelination without added NRG (p = 0.27), but in
the presence of NRG it reduced myelination by 82% (p = 561026;
Figure 7A) to only 26% of the level seen without NRG
(significantly lower, p = 0.0024). Thus, unlike the activity-independent mode of myelination, the activity and NMDA receptordependent myelination induced by NRG is dependent on integrin
function.
PLOS Biology | www.plosbiology.org
8
December 2013 | Volume 11 | Issue 12 | e1001743

10.
NRG, NMDARs, and Activity Regulate CNS Myelination
by NRG. Furthermore, the influence of NMDA receptors on
myelination is not limited to myelination occurring during normal
development, – because remyelination of demyelinated axons in
the cerebellar peduncle in vivo also depends on NMDA receptor
activation (Figure 8).
Adding NRG increases 6-fold the NMDA-evoked currents in
oligodendrocyte precursor cells and in differentiated oligodendrocytes, but does not significantly alter NMDA-evoked currents in
DRG neurons, nor NMDA- or kainate-evoked currents in any
other cell type in the culture (Figure 5), nor the action potential
firing of DRG axons (Figure 4). We therefore attribute the NRGinduced activity dependence of myelination to a potentiation of
NMDA receptor signalling in oligodendrocyte lineage cells,
making the cells more sensitive to axonal activity. This potentiation may largely reflect a decreased protein level of NR3A
subunits (Figure 6). However, the 6-fold increase in NMDA
receptor-mediated current that we find is larger than the 2.8-fold
increase reported in neurons when NR3A is knocked out [32].
Consequently, because of possible obscuring of changes in
oligodendrocyte protein levels by neuronal NMDA receptor
protein in the cocultures, we cannot rule out a contribution to
the increase in NMDA-evoked current from alterations of the
levels of other NMDA receptor subunits in oligodendrocyte
lineage cells, of which upregulation of NR2C [21] and/or NR2B
[30] subunits are the most likely candidates.
In the presence of NRG, blocking action potentials reduces
myelination less than does blocking NMDA receptors (Figure 2A),
despite the fact that it is presumably action potentials that release
the glutamate that activates the NMDA receptors. Thus, in NRG,
TTX reduces myelination to a value close to that occurring
without NRG present (Figures 2A and S5A). This may reflect
action potential evoked glutamate release being needed for NRG
to increase NMDA receptor currents, so that the switch to NMDA
receptor-dependent myelination does not occur in the absence of
action potentials. This is likely since NMDA receptor activation is
needed both for removal of NR3A subunits from the surface
membrane [56] and for NRG to upregulate NR2C subunits in
neurons [21]. Consistent with this, blocking the glutamate site on
NMDA receptors with D-AP5 reduced myelination in NRG less
than did MK-801 or 7-chlorokynurenate, which block at other
sites on NMDARs (Figure 2A), and in pure OPC cultures, NRG
downregulated NR3A only if glutamate was also added
(Figure 6D). The inhibitory effect of NBQX in NRG in
Figure 2A may similarly reflect AMPA/kainate receptor activity
maintaining neuronal firing, which evokes the glutamate release
needed to upregulate NMDA currents [21,56], or could reflect
AMPA/kainate receptor-mediated depolarisation increasing current flow through NMDA receptors [18].
The exact sequence of signalling steps in oligodendrocytes that
upregulates NMDA receptor currents, and thus speeds, increases,
and confers activity-dependence upon myelination, remains to be
defined, although we have shown that Akt, integrin b1, CREB,
and EGR-1 are involved (Figure 7). Since NRG activates Akt
(Figure 7B) and Fyn [30] and these kinases are known to alter
NMDA receptor subunit expression and trafficking [30,31] and to
promote myelination [16,46,57], it is an attractive idea that NRG
may initially activate Akt and/or Fyn, and that this action is
potentiated by integrins (Figure 7A) [34,35,57].
Our results help resolve three controversies in the field. The
first concerns the role of NRG in CNS myelination. Decreasing
NRG or ErbB function reduces myelination [5–7], and in the
prefrontal cortex, NRG’s control of myelination is regulated by
social interactions [58]. However, knocking NRG or ErbB out
had no effect on myelination, even though overexpressing NRG
myelination may still occur [9] not only via the activityindependent mode of myelination (Figure 2A) but also because
of compensatory BDNF signalling (see Discussion).
Remyelination in Vivo Is NMDA Receptor Dependent
Our demonstration of NRG- and NMDAR-dependent myelination of axons by oligodendrocytes raises the question of whether
this same mechanism operates during remyelination after white
matter damage. A lack of NRG signalling has been suggested to
result in poor remyelination in multiple sclerosis [51,52]. The data
above suggest this may reflect a lack of NRG-dependent
upregulation of NMDA receptors in oligodendrocyte lineage cells,
but NMDA receptor deletion or block has been variously reported
to either have no effect on loss of myelin in the experimental
autoimmune encephalomyelitis model of multiple sclerosis [24] or
to delay remyelination after cuprizone demyelination [25]. We
therefore tested whether successful remyelination is dependent on
NMDA receptor activation, in OPCs that are recruited to
remyelinate axons in a focal toxin-induced demyelinated lesion
in the rat caudal cerebellar peduncle. This demyelination model
provides successful spontaneous remyelination that occurs with a
clear temporal separation from the acute demyelination [53].
An intracerebral implanted cannula, connected to an osmotic
minipump, infused into the lesion either 0.9% saline or 50 mM
MK-801 (at a flow rate of 0.11 ml/h) from the 3rd day postlesion
(the timepoint when OPCs enter the lesion, [54]) until the animal
was sacrificed at 21 d postlesion. Analysis of semithin sections
stained with toluidine blue showed that there was no difference in
lesion size (saline, 0.2860.04 mm2; MK-801, 0.2260.04 mm2,
p = 0.28) nor axon density (saline, 50,80062,100 axons/mm2;
MK-801, 47,50061,900 axons/mm2, p = 0.27) between the
conditions, but a blinded analysis ranking remyelination revealed
that blocking NMDA receptors with MK-801 significantly
inhibited remyelination (p = 0.036; Figure 8A–C). Moreover, at
the ultrastructural level, it was clear that fewer axons were
remyelinated when NMDA receptors were blocked (p = 0.0012;
Figure 8D–F), and the g-ratio (the ratio of axon diameter to
outside diameter of the myelin) of remyelinated axons was higher
in MK-801– compared to saline-infused lesions (p = 0.01;
Figure 8G–I), showing that the myelin was thinner. Thus, efficient
remyelination depends on activation of NMDA receptors.
Discussion
The speeding of action potential propagation produced by
myelination of axons is crucial for CNS function. We have shown
that, in the absence of added NRG, myelination takes place by a
mechanism that is independent of neuronal activity and glutamate
release, because it is unaffected by blocking action potentials or
ionotropic glutamate receptors. With NRG added, myelination is
accelerated and increased, this effect is dependent on action
potentials, and blocking NMDA receptors greatly reduces
myelination (to well below the level seen in the absence of added
NRG; Figures 1 and 2). Thus, the key result of this article is that
NRG does not just speed and increase myelination; it produces a
switch in the mechanism of myelination, from a default
programme that is independent of neuronal activity (which allows
oligodendrocytes to ensheath even fixed axons [12]) to a
programme that depends on activation of NMDA receptors in
oligodendrocyte lineage cells, presumably by glutamate released
from active axons [14–18,55] (although we cannot rule out a
contribution of glutamate release, either tonic or activity-induced,
from other cells). This switch implies a suppression of the default
programme when the activity-dependent programme is activated
PLOS Biology | www.plosbiology.org
10
December 2013 | Volume 11 | Issue 12 | e1001743

12.
NRG, NMDARs, and Activity Regulate CNS Myelination
Figure 7. Signalling underlying the effects of NRG. (A) Effect of blocking integrin function with an antibody to the b1 subunit (b1) in the
absence and presence of NRG (number of experiments shown on bars). ANOVA across all conditions gave p,0.0001. The p values above each bar
compare with control. (B–C) Western blot of control (Con) and NRG cocultures (numbers of co-cultures shown on bars), in the absence and presence
of MK-801, for phosphorylated (pAkt, pERK) and total Akt and ERK, with level of phosphorylated enzyme normalized to total enzyme in bar charts
below. (D) Cocultures labelled for nuclei with DAPI, and with antibodies to Nkx2.2 (for late OPCs/immature oligodendrocytes) and pCREB (arrows
show cells expressing both). (E) Percentage of cells expressing and not expressing Nkx2.2, which label for pCREB (in 29 control and 29 NRG fields of
view, including 26,261 control and 21,518 NRG cells). (F) Change in immediate early gene expression mRNA level (by qPCR) in NRG-treated cocultures,
normalized to control levels. (G) Effect of BDNF and MK-801 on myelination. One-way ANOVA gave p,0.0001 (the potentiation of myelination by
BDNF here cannot be directly compared with that for NRG in Figure 2A because the experiments were not done on the same set of cocultures). (H–I)
Western blot of control (Con) and BDNF cocultures, for phosphorylated (pAkt, pERK) and total Akt and ERK, with level of phosphorylated enzyme
normalized to total enzyme in bar charts below. (J) Western blots of control (Con) and BDNF-treated cocultures for NR1 and NR3A, with densitometric
quantification of subunit protein levels in bar graph below. The p values over single bars compare with control (Con). The p values are from Holm–
Bonferroni corrected t tests in (A and G), Dunnett’s post hoc tests in (B and C), and Student’s t tests in (H–J). Number of cultures are shown on bars.
doi:10.1371/journal.pbio.1001743.g007
predominantly activity dependent or independent may depend on
the amount of NRG or BDNF (or other molecules [69,70]) being
expressed or released [21,71]. In this context it is worth noting
that, although we have discussed our results as reflecting the
presence or absence of NRG, in the absence of added NRG,
there will be some level of endogenous NRG release, and it may
be better to think of whether the NRG level is low or high as
determining the main myelination mode that occurs. It is also
possible that the switch from one mode to the other occurs
gradually as the NRG level rises.
The existence of two alternative myelination programmes
regulated by NRG level, independent of, and depending on,
action potentials and NMDA receptors, may reflect the evolutionary importance, but also the metabolic cost, of myelination.
One can speculate that early in development it may be important
to myelinate whatever axons are present, irrespective of their
impulse traffic. However, once a significant density of axons is
present, because myelination involves considerable investment by
the oligodendrocyte in lipid production [72], it is more efficient for
myelination to be focussed on axons that have a high impulse
traffic, rather than on inactive axons. The NRG- and NMDA
receptor-dependent mode of myelination may dominate later in
development, since late myelination in the prefrontal cortex
depends on NRG signalling [58] and adult remyelination depends
on NMDA receptor activation (Figure 8).
NRG is a susceptibility gene for schizophrenia [22,73], and
NMDA receptors are also implicated in this disease [74]. NRG
affects NMDA receptor expression in the grey matter [21,22],
where the defect underlying schizophrenia is usually assumed to
occur. However, correct myelination is essential for normal
cognitive function, and the interaction of NRG and NMDA
receptors to control myelination allows us to speculate that there
could perhaps be a white matter explanation for the linkage of
NRG and NMDA receptors to schizophrenia. Furthermore, NRG
expression is reduced in multiple sclerosis lesions [52], and adding
NRG has been suggested to promote remyelination in a mouse
model of multiple sclerosis [51], suggesting that NRG- and
NMDA receptor-dependent remyelination may be important after
pathology. Consistent with this we have shown that, in vivo in the
cerebellar peduncle, successful remyelination is reduced when
NMDA receptors are blocked (Figure 8) and similar results have
been found for remyelination in the corpus callosum [25].
Promoting NRG- and NMDA receptor-dependent myelination
may, therefore, be a useful therapeutic strategy for increasing CNS
remyelination in disease.
increased myelination [9]. Our data predict that myelination can
appear to be unaffected when NRG-ErbB signalling is abolished,
for three reasons. First, the activity-independent mode of
myelination (Figure 2A) should still occur. Second, when
myelination levels are high, as may be the case in vivo, NRG
only modestly increases myelination (Figure 2D), yet it still makes
myelination become very highly dependent on neuronal activity
releasing glutamate to activate NMDA receptors (Figure 2E).
Third, our data indicate redundancy in the growth factors that
switch oligodendrocytes between the two myelination modes.
Both NRG and BDNF alter NMDA receptor expression in
oligodendrocyte lineage cells, induce Akt activation but not Erk
activation (although promotion of myelination by BDNF can also
involve Erk activation [59]), and promote myelination, and both
switch oligodendrocytes to the NMDA receptor-dependent mode
of myelination (Figures 2A and 7G). Since not only ErbB
receptors for NRG but also TrkB receptors for BDNF (including
full length receptors [49,50,60–62]) can be expressed by
oligodendrocytes, our data suggest that the failure of NRG or
ErbB knockout to affect CNS myelination may, in part, reflect
another growth factor (BDNF) and its receptor (TrkB) acting to
replace the NRG/ErbB system.
The second controversy concerns the role of NMDA receptors
in CNS myelination. Deleting the NMDA receptor NR1 subunit
from oligodendrocyte lineage cells has been reported to have no
effect on myelination [23,24], yet activation of NMDA receptors
by glutamate released from active axons is reported to promote
MBP translation and myelination [16]. Interestingly, in another
oligodendrocyte-specific NR1 knockout, deletion of NMDA
receptors slowed myelination in the optic nerve [63], consistent
with the acceleration of myelination that we observe when NMDA
receptor currents are increased in the presence of NRG
(Figure 2C). This effect of NMDA receptors was proposed to be
caused by NMDA receptor activation upregulating glucose
transporters in oligodendrocyte lineage cells (as was previously
shown to occur in an Akt-dependent manner in neurons; [64]) to
provide energy for myelination [63].
The third controversy concerns whether myelination is activity
dependent or independent. Rearing rodents in the dark from
birth, or injecting TTX into the optic nerve, can reduce or
alternatively have no effect on myelination [10,65–67]. Similarly,
electrical activity [10,16], or application of factors it releases [11],
can promote myelination, and blocking neuronal activity with
TTX in myelinating cortical cultures reduced myelination [10],
whereas in spinal cord cultures TTX did not affect myelination
[68]. Presumably, these differences may reflect the existence of
the two alternative myelination programmes that we have
characterised (whereby in the absence of added NRG, TTX
has no effect on myelination, but in NRG-treated cultures, it
reduces myelination by 50%; Figure 2A). Whether myelination is
PLOS Biology | www.plosbiology.org
Material and Methods
DRG-Oligodendrocyte Cocultures
These were made as described previously [8]. Briefly, DRG cells
from E14–E16 rats were cultured for 2 wk, and then cultured
12
December 2013 | Volume 11 | Issue 12 | e1001743

13.
NRG, NMDARs, and Activity Regulate CNS Myelination
Figure 8. Remyelination is dependent on NMDA receptor activation. (A–B) Semithin sections of lesioned caudal cerebellar peduncle (CCP),
21 d postlesion, infused with saline (A) or MK-801 (50 mM; B) for 18 d. The dotted white lines mark the lesion edge. (C) Ranking of remyelination; each
symbol represents one animal. Higher ranks represent more remyelination. The p value from Mann–Whitney U test. (D, E) Higher magnification
picture shows that fewer axons are remyelinated in lesions treated with MK-801 (remyelinated axons are coloured green). (F) Mean percentage of
axons remyelinated averaged over 20 areas in each of three lesions for each condition. (G) Specimen images of a normal myelinated axon, a
demyelinated axon, and remyelinated axons in lesions infused with saline or MK-801. (H–I) Mean g-ratio of all axons, and mean g-ratio at all
diameters, are higher with MK-801 present (student t test, n = 3).
doi:10.1371/journal.pbio.1001743.g008
oligodendrocyte precursor cells (OPCs) from P0–P2 rats were
plated on top of them [8]. One coverslip was analysed for each
drug condition per dissection. Three weeks later, the cocultures
were fixed, labelled, and images were taken of each coverslip to
assess myelination (see detailed description below) [8]. NRG (Lab
Vision) or BDNF (R&D systems) and receptor blockers (Tocris)
were applied with the OPCs (except when TTX was added 3 d
later than the NRG and OPCs) and were included in medium
changes twice per week thereafter. Culture medium contained
0.8 mM MgCl2 and 0.4 mM glycine, but no added glutamate.
PLOS Biology | www.plosbiology.org
DRG Cultures
As described above, DRG cells from E14–E16 rats were
cultured for 2 wk, then put in myelination medium (as for
cocultures but lacking OPCs), for 3 wk, with medium changes
twice per week thereafter.
Oligodendrocyte Cultures
These were as described previously [8]. Briefly, purified oligodendrocyte precursors were obtained with minor modifications
13
December 2013 | Volume 11 | Issue 12 | e1001743

14.
NRG, NMDARs, and Activity Regulate CNS Myelination
[36] of the method of McCarthy and de Vellis [75]. They were
cultured in myelinating medium (the same as for the cocultures)
for 6 d, either with or without 20 min 100 mM glutamate
stimulation every day for the last 5 d in vitro.
counted the fraction of oligodendrocytes that myelinated, by
averaging over all the sampled images, then the results were
broadly similar, except that NMDA receptor block in control
conditions produced a reduction of myelination of borderline
significance (Figure S3D).
Quantification of Myelination
Electrophysiology
For each coverslip, 30 randomly located images were taken of
MBP and NF staining (using either a 106 objective, with a field of
view of 843 mm6636 mm, or a 206 objective, with a field of view
of 709 mm6530 mm). Within each image, myelination was
quantified by counting the number of myelinating MBP-positive
oligodendrocytes (Figure 1B,C) as a percentage of the total
number of MBP-positive oligodendrocytes [8]. The density of
axons was expressed as the percentage of the same area occupied
by NF (calculated from a binarised NF image using ImageJ
software, version 1.34s). There was no correlation between
oligodendrocyte density and axon density. The fraction of
myelinating cells was plotted against the axon density for the
different images, and fitted assuming a linear dependence [8] of
fraction of oligodendrocytes myelinating (F) on axon density (D),
F ~A:ðD{K Þ,
Cells were whole-cell patch-clamped [14,18] at room temperature (21–24uC) in cocultures 3 wk after the OPCs were added.
Electrodes contained solution comprising (mM) either 126 CsCl, 4
NaCl, 10 HEPES, 5 EGTA, 4 MgATP, 0.5 Na2GTP, 12
phosphocreatine, 2 K-Lucifer yellow, pH set to 7.3 with CsOH
(ECl = 0 mV), or 130 Cs-gluconate, 4 NaCl, 0.5 CaCl2, 10
HEPES, 10 BAPTA, 4 MgATP, 0.5 Na2GTP, 2 K-Lucifer yellow,
pH set to 7.3 with CsOH (ECl = 288 mV). Series resistance was
5–20 MV, and electrode junction potentials were compensated.
Cultures were superfused at 2461uC with HEPES-buffered
solution containing (mM) 144 NaCl, 2.5 KCl, 10 HEPES, 1
NaH2PO4, 2.5 CaCl2, 10 glucose, 0.1 glycine (to coactivate
NMDA receptors), 0.005 strychnine (to block glycine receptors),
pH set to 7.4 with NaOH, bubbled with 100% O2. Cells were
identified (Figures 4A and 5A–F) by their postrecording dye-fill
morphology [14,18], confirmed by antibody labelling against the
proteoglycan NG2 to identify oligodendrocyte precursors (17/17
tested labelled for NG2), against CNPase (11/11), MBP (10/10),
MOG (4/4), or GalC (11/11) for differentiated oligodendrocytes,
against GAD 65/67 for interneurons (21/21), against NF 160/
200 (13/13) for DRG neurons, against GFAP for astrocytes (11/
11), and against SCIP for satellite cells (10/10). In addition we
checked that each recorded cell had the electrophysiological
properties expected for its class: specimen responses to voltage
steps from the resting potential are shown for each cell class in
Figure S4. OPC morphology cells, including those confirmed as
being OPCs by virtue of their labelling with antibody against
NG2, fell into two classes [14] with (57% of cells) and without
(43% of cells) voltage-gated Na+ current (Figure S4A–D), and
had a steady-state input resistance of ,800 MV. Mature
myelinating oligodendrocytes had a roughly ohmic and timeindependent I-V relation in the physiological range (Figure S4E),
with a much lower input resistance of ,160 MV. Interneurons
and DRG neurons both showed a voltage-gated Na+ current on
depolarization, which was too large to clamp well, so the I-V
relations showed current oscillations reflecting uncontrolled
action potentials occurring (Figure S4F,G). Both satellite cells
and astrocytes showed roughly ohmic and time-independent I-V
relations in the physiological range (Figure S4H,I) with a mean
input resistance near the resting potential of ,900 and ,15 MV,
respectively.
ð1Þ
where K is a free constant and the slope A is the measure of
myelination plotted in Figure 2, Figure S2A, Figure S3A, and
Figure 7. Fitting data in control and NRG conditions gave values
for K that were not significantly different (12.961.3%, n = 32
experiments, for control, and 12.661.8%, n = 27, for NRG;
p = 0.88). This quantification of myelination gave results similar to
those obtained using other assumed dependencies of fraction of
oligodendrocytes myelinating (F) on axon density (D) as follows:
(1) A linear dependence above a threshold density:
F ~0 for DƒDthreshold and
F ~A:ðD{Dthreshold Þ for DwDthreshold :
ð2Þ
Fitting data in control and NRG conditions with Dthreshold, a free
parameter gave thresholds that were not significantly different
from Dthreshold = 17% (17.961.8%, n = 32, p = 0.62 for control, and
17.262.4%, n = 27, p = 0.93 for NRG), so Dthreshold was set to 17%;
we refitted the data, and used the value of A as a measure of
myelination (Figure S2B, Figure S3B).
(2) A power law:
F ~A:DN :
ð3Þ
Fitting data in control conditions or in the presence of NRG with
N a free parameter gave best fit powers not significantly different
from N = 2 (2.0860.14, n = 32, p = 0.57, and 2.1060.16, n = 27,
p = 0.54, respectively), so we fixed the power at 2, re-fitted the
data, and then used the amplitude (A) of the best fit curve as the
measure of myelination (Figure S2C and Figure S3C).
Synaptic Current Analysis
A synaptic current was defined to occur if its amplitude was .3
times the standard deviation of the baseline current noise and its
10%–90% decay time was longer than its rise time. Events were
detected and analysed with pClamp 10 (Axon Instruments).
Irrespective of the quantification used, the bar charts in different
conditions (Figure S3A–C) gave the same results: blocking action
potentials or NMDA receptors in control conditions had no effect
on myelination, NRG significantly increased myelination, and in
the presence of NRG blocking action potentials produced a
significant reduction of myelination, while blocking NMDA
receptors reduced myelination so strongly that it was significantly
reduced below the control level with no NRG. If, instead of fitting
the dependence of myelination on local axon density, we simply
PLOS Biology | www.plosbiology.org
Immunohistochemistry
Cultures were fixed at 21uC for 20 min in 4% PFA and
incubated for 1 h in 0.1% Triton X-100, 10% goat serum in
phosphate-buffered saline at 21uC, then with primary antibody at
21uC for 2 h or for overnight at 4uC, and then for 1 h at 21uC
with secondary antibody. Primary antibodies were guinea pig
NG2 (a kind gift from W.B. Stallcup, 1:100), rabbit NG2
(Chemicon, 1:300), rabbit Olig2 (a kind gift from D. Rowitch,
14
December 2013 | Volume 11 | Issue 12 | e1001743

15.
NRG, NMDARs, and Activity Regulate CNS Myelination
C.D. Stiles & J. Alberta, 1:20,000, or Chemicon, 1:1,000), rabbit
Caspr (a kind gift from D. Colman & J. Huang, 1:500), rabbit
SCIP (a kind gift from J.R. Bermingham, 1:100), rabbit GalC
(Sigma, 1:100), mouse NF 160/200 (Sigma, 1:1,000), rat MBP
(Serotec, 1:100), mouse MOG (Sigma, 1:100), mouse CNPase
(Sigma, 1:100), rabbit GAD 65/67 (Chemicon, 1:100), rabbit
GFAP (Dako, 1:500), chicken P0 (Aves Labs, 1:500), mouse
Nkx2.2 (DSHB, 1:120), CD11b (Serotec, 1:50), and rabbit pCREB
(Cell Signalling, 1:50). Alexa 488–conjugated isolectin B4 (Invitrogen, 1:100) was used to label microglia. DAPI (Sigma, 20 mM) was
used to label nuclei. Secondary antibodies (goat) were for rabbit
(Molecular Probes, 1:1,000), rat IgG (Molecular Probes, 1:1,000),
mouse IgG (Molecular Probes, 1:1,000), chicken (Jackson Lab,
1:1,000), and guinea pig (Jackson Lab, 1:100).
converted to cDNA using a SuperScript First-Strand Synthesis
System for RT-PCR (Invitrogen). qPCR was performed with a
QuantiFast SYBR Green PCR Kit (QIAGEN) using a LightCycler
480 II. Samples were normalised to GAPDH levels using
commercially available QuantiTect Primers (QIAGEN) for analysis.
Induction of Focal Demyelination
Female Sprague-Dawley rats aged 9–12 wk of age (200–225 g)
were used for remyelination studies, which were performed in
compliance with UK Home Office regulations. Focal demyelination was induced unilaterally by stereotaxically injecting 4.0 ml of
0.01% ethidium bromide (w/v) in saline into the caudal cerebellar
peduncle (CCP) (as described previously [53]). For continuous
local delivery of MK-801 (50 mM, Tocris, in 0.9% saline) or 0.9%
saline (Vetivex) into the demyelinated lesion, an osmotic minipump
with a reservoir volume of 100 ml and a flow rate of 0.11 ml/h (Alzet
Micro-Osmotic Pumps, model 1004, DURECT Corporation) was
attached through a vinyl tube spacer (Plastics One Inc., Roanoke,
Virginia) to a 30 gauge (6.5 mm) cannula implanted just above the
lesion. The length of the tube was cut to 2.3 cm, to ensure that drug
delivery into the lesion did not occur until the 3rd day postlesion (the
start of the OPC recruitment stage [54]). The minipump was placed
subcutaneously, and the cannula was fixed to the skull with
cyanoacrylate gel adhesive (applied under the base of the cannula
head before insertion), as well as two anchoring screws and dental
acrylic cement (a 1:1 volume mix of Paladur powder and liquid;
Heraeus Kulzer). Rats were randomly assigned to treatment (50 mM
MK-801) or control groups (0.9% saline infusion). Animals were
sacrificed 3 wk after lesion induction.
Western Blots
For protein analysis, cultures were scraped from 22 mm
coverslips, 6 well plates, or 10 cm dishes and lysed mechanically
in solution containing 0.1 M phosphate buffered saline (PBS), 10%
or 20% (w/v) sucrose and Halt protease, and phosphatase
inhibitor cocktail (Thermo Scientific). Protein content was
determined by Bradford assay and quantified using a standard
curve obtained from Quick Start BSA protein standards (Bio-Rad).
Equal amounts of protein (8 or 10 mg) from samples were resolved
on 4–%12% NuPage Novex Bis-Tris mini gels (Invitrogen), with
prestained molecular weight protein standards (Bio-Rad). Proteins
were transferred to a nitrocellulose membrane (0.45 mm, GE
Healthcare) using a wet transfer system. Nitrocellulose membranes
were blocked for 1 h at room temperature with 3% BSA in PBS
with 0.1% Tween-20 (PBS-T). Immunoblots were then incubated
overnight at 4uC with goat anti-Akt (Santa Cruz, 1:1,000), rabbit
anti-phosphorylated-AktSer473 (Cell Signalling, 1:1,000), rabbit
anti-phosphorylated-ERK1/2Thr202/Tyr214
(Cell
Signalling,
1:1,000), mouse anti-ERK1/2 (Cell Signalling, 1:2,000), rabbit
anti-MBP (Sigma, 1:3,000), mouse anti-NR1 (Millipore, 1:1,000),
rabbit anti-NR2A (Millipore, 1:1,000), rabbit anti-NR2B (Abcam;
1:1,000), rabbit anti-phosphorylated NR2BTyr1472 (Millipore,
1:1,000), rabbit anti-NR2C (Millipore, 1:1,000), rabbit antiphosphorylated NR2CS1096 (a kind gift from Katherine W. Roche,
NINDS), rabbit anti-NR3A (Millipore, 1:500), rabbit anti-NR3B
(Millipore, 1:300), rabbit anti-NR2D (Abcam, 1:300), or mouse
anti-b-actin (Sigma, 1:100,000) in 3% BSA in PBS-T. This was
followed by incubation with the secondary horseradish peroxidiselinked anti-rabbit, anti-mouse, or anti-goat antibodies (1:1,000,
Dako) for 1 h at room temperature in 3% BSA in PBS-T.
Immunoreactive proteins were visualised with enhanced chemiluminescence (GE Healthcare). When phosphorylated and total
amounts of the same protein were measured, this was done by first
probing with the antibody to the phosphorylated protein, then
stripping the membranes to remove the antibody, and reprobing
with antibody recognizing both phosphorylated and unphosphorylated forms. Stripping membranes was performed with
50 mM dithiothreitol, 50 mM Tris (pH 6.8), and 2% SDS at 70uC
for 30 min and washing three times in PBS-T before blocking and
incubating with the primary antibody. Densitometric analysis was
conducted using ImageJ gel analysis software (version 1.43u). For
each sample, pAkt, pERK, pNR2B, and pNR2C signals were
normalised to the total levels of their respective proteins present.
The levels of nonphosphorylated proteins were normalized against
b-actin.
Electron Microscopy
Co-cultures were fixed at room temperature for 1 h in PBS
containing 2.5% glutaraldehyde (Agar Scientific) and 0.72 mM
CaCl2. The cocultures were scraped off the coverslips, placed into
1.5 ml tubes with 250 ml of PBS, and centrifuged at 800 g for
3 min. For lesion samples, animals were perfusion fixed with 4%
glutaraldehyde (Agar Scientific) in phosphate buffer containing
0.72 mM CaCl2. All samples were then left to fix in 2% osmium
tetroxide (Oxkem Ltd) in phosphate buffer at 4uC overnight. This
was followed by dehydration in 70% ethanol for 15 min, 95%
ethanol for 15 min, and 100% ethanol for 3610 min, then the
tissue was placed in propylene oxide for 2615 min, and then left
for at least 3 h in a 50%/50% mixture of propylene oxide and
resin mix (containing by volume: 49% TAAB embedding resin,
33% DDSA, 16% MNA, 2% DMP-30; TAAB Laboratories
Equipment Ltd.). They were then transferred to 100% resin mix
overnight. New resin was made and the samples left in it for at
least 6 h before being placed in embedding capsules and further
incubated at 60uC for 15–24 h until the resin was solid. Embedded
samples were cut in 90 nm sections on an ultramicrotome
(Reichert Ultracut E) with a diamond knife (Diatome) and
visualised using a Transmission Electron Microscope (Hitachi
H600). Images were developed on electron microscope film 4489
(Kodak), then scanned at high resolution (12,800 dpi612,800 dpi),
and analyzed with ImageJ software, version 1.34s.
Histological Analysis of Demyelination and
Remyelination
Animals were perfused with 4% glutaraldehyde (in phosphate
buffer with 0.72 mM CaCl2), and the brains were immersion fixed
in 4% glutaraldehyde for 7 d. Tissue blocks encompassing the
caudal cerebellar peduncle were cut as detailed previously [53].
While maintaining their correct orientation and sequence, blocks
Quantitative RT-PCRs
RNA was extracted from DRG-OPC cocultures 21 d after
addition of OPCs using an RNeasy mini kit (QIAGEN). RNA was
PLOS Biology | www.plosbiology.org
15
December 2013 | Volume 11 | Issue 12 | e1001743

16.
NRG, NMDARs, and Activity Regulate CNS Myelination
were further fixed in 2% osmium tetroxide (Oxkem Ltd.),
dehydrated in increasing concentration of ethanol, and embedded
in resin (TAAB Laboratories). One micron sections were cut and
stained with toluidine blue. In these sections, remyelinated axons
can be easily distinguished from normally myelinated axons
outside the lesion by the thinness of the myelin sheath. Within the
lesion, remyelinated axons can be distinguished from demyelinated axons because the former possess myelin sheaths recognizable as a dark staining rim around the axon.
together with the myelination parameter (A) derived from the fit
for the four conditions for this particular set of coverslips. The
dependencies assumed were (A) the linear relation: F = AN(D–K),
where K is a free constant, as used previously [8]; (B) the 17%
threshold-linear relation: F = 0 for D#17% and F = AN(D–17%)
for D.17%; (C) the power 2 relation: F = AND2.
(EPS)
Figure S3 Myelination in various conditions quantified
in different ways. Graphs show the myelination parameter for
the data analysed for Figure 2A of the main text derived from
different assumed dependencies of myelination on axon density. (A)
Linear relation with intercept not fixed. For no NRG, ANOVA
showed no significant differences across bars (p = 0.72); for with
NRG, ANOVA showed significant differences across bars
(p,0.0001). (B) Threshold-linear relation with threshold of 17%.
ANOVA gave p = 0.77 for no NRG and p,0.0001 for with NRG.
(C) Power 2 relation. ANOVA gave p = 0.76 for no NRG and
p,0.0001 for with NRG. (D) Fraction of oligodendrocytes myelinating, averaged across all images. ANOVA gave p = 0.096 for no NRG
and p,0.0001 for with NRG. For all panels the p values, comparing
data in no NRG with control, and comparing data in with NRG with
NRG alone, were from Holm–Bonferroni tests.
(EPS)
Statistics
Data are mean 6 s.e.m. The p values are from ANOVA and
post hoc Student’s two-tailed t tests. For multiple comparisons p
values are corrected using a procedure equivalent to the Holm–
Bonferroni method (for N comparisons, the most significant p value
is multiplied by N, the 2nd most significant by N–1, the 3rd most
significant by N–2, etc.; corrected p values are significant if they are
less than 0.05) or Dunnett’s post hoc test. Normality of data was
assessed using Shapiro–Wilk tests, and nonparametric Kruskal–
Wallis and Mann–Whitney tests, which do not assume data follow
a normal distribution, gave the same conclusions for significant
and nonsignificant differences in all cases. For Figure 2, when the
ANOVA gave an insignificant p value (.0.05), we used the more
conservative Student’s two-tailed t test without correction for
multiple comparisons to give a lower limit to the p value for post
hoc comparisons (i.e., the differences among the control myelination data in Figure 2A are even less significant than is indicated by
the p values).
Figure S4 Electrophysiological characteristics of the
cells in the cocultures. Specimen responses to voltage steps
in 20 mV increments from a holding potential of 264 mV (to a
most negative potential of 2104 mV and a most positive potential
of +16 mV, for (A) an OPC with voltage-gated Na+ current (NaV),
(B) an OPC without NaV, (C) the OPC in (A) on a faster time
scale, (D) the OPC in (B) on a faster time scale, (E) a mature
oligodendrocyte, (F) an interneuron, (G) a DRG neuron, (H) a
satellite cell, and (I) an astrocyte. In (F and G) transient currents
generated by unclamped action potentials are visible.
(EPS)
Supporting Information
Characterization of myelination in the cocultures. (A) Electron microscopic image of myelination in the
cocultures showing formation of compact myelin. (B) Labelling of
coculture for Caspr shows formation of axon-oligodendrocyte
junctions at the end of internodes. Note that some axons have
single internodes so that only one Caspr-labelled region is visible at
a heminode (open arrows), while some have multiple adjacent
internodal segments, so that two Caspr-labelled regions are visible
at nodes (filled arrows). (C) Enlarged view of a node of Ranvier
labelled for Caspr. (D) DRG cells with no added OPCs show no
myelination, ruling out the possibility of myelination by Schwann
cells or precursors added with the DRG cells. Blue is DAPI to label
nuclei, red is NF 160/200, and green is MBP (which is absent). (E)
Myelination is by MBP-expressing OPCs and not P0-expressing
Schwann cells. Mauve is NF, green is MBP, and red is P0 protein
(a component of myelin made only by Schwann cells, which is
absent). (F) The processes of DRG cells but not of interneurons
become myelinated. Red is NF, green is MBP, and white is the
GABA synthesizing enzyme glutamate decarboxylase (GAD 65/
67). (G) Western blots of control (Con), NRG-treated and BDNFtreated cocultures for MBP (b-actin acts as a loading control), and
densitometric quantification of subunit protein levels (normalized
to b-actin and then to control). (H) Change in myelin gene mRNA
level (by qPCR) in NRG-treated cocultures normalized to control
levels. All p values are from one-sample Student’s t tests. Numbers
of cultures are shown on bars.
(EPS)
Figure S1
Figure S5 Details of the signalling pathways. (A) Mean
myelination parameter of cocultures treated with NRG with TTX
either added at the same time as NRG (TTX) or 3 d later (TTX
3 d). ANOVA showed significant differences across all bars
(p = 0.025); the p values above bars are for comparison with
NRG alone. (B–C) Western blot of control (Con) and NRGtreated pure DRG cultures (cultured as for myelinating OPCDRG cocultures), for phosphorylated (pAkt, pERK) and total Akt
and ERK, with the level of phosphorylated enzyme normalized to
total enzyme in the bar charts below. The p values are from
Student’s t tests; numbers of experiments are shown on bars.
(EPS)
Acknowledgments
We thank W. Stallcup for NG2, J.R. Bermingham for SCIP, D. Colman
and J. Huang for Caspr, K.W. Roche and B.S. Chen for pNR2C
antibodies, Mike Peacock for EM assistance, and Kirsten Caesar, Claudia
Eder, Alasdair Gibb, Nicolas Granger, Nicola Hamilton, Kristjan Jessen,
Ilias Kazanis, Karolina Kolodziejczyk, Lesley Probert, Angus Silver, and
Jing-Wei Zhao for comments on the manuscript.
Author Contributions
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: RTK DA Cff
IL AL JHS. Performed the experiments: IL AL JHS KAE MS KV ZW
HOBG RTK. Analyzed the data: IL AL JHS KAE MS KV ZW HOBG
DA RTK. Contributed reagents/materials/analysis tools: Cff DA RTK.
Wrote the paper: IL AL RJMF Cff DA RTK.
Figure S2 Quantification of myelination. Each set of
graphs shows the same data (from Figure 1C–F of the main text)
in control conditions, control+MK-801, NRG and NRG+
MK-801, best fit with an assumed dependence of myelination
(fraction of oligodendrocytes myelinating, F) on axon density (D),
PLOS Biology | www.plosbiology.org
16
December 2013 | Volume 11 | Issue 12 | e1001743